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Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols
Published on: October 18, 2016
Cell-associated IL-8 in human blood monocytes: analysis by flow cytometry
S Fujishima1, H Nakamura, Y Waki
1Department of Medicine, School of Medicine, Keio University, Tokyo, Japan.
Insights
Cell-associated interleukin-8 (IL-8) was not detected on the surface of lipopolysaccharide-stimulated monocytes. This suggests that cell-surface IL-8 likely does not play a significant physiological role in immune responses.
Area of Science:
- Immunology
- Cell Biology
Background:
- Cell-associated cytokines, like IL-1 and TNF, are biologically active on cell surfaces.
- Extracellular IL-8 is a key neutrophil chemoattractant, but cell-associated IL-8 remains understudied.
Purpose of the Study:
- To investigate the presence and localization of intracellular and cell-surface IL-8 in human blood monocytes.
- To predict the in vivo biological activity of cell-associated IL-8.
Main Methods:
- Flow cytometry was used to analyze intracellular and cell-surface IL-8 in monocytes.
- Mononuclear cells were fixed and either left untreated or permeabilized with saponin.
- Immunohistochemical analysis confirmed differential cytokine localization.
Main Results:
- Lipopolysaccharide (LPS) stimulation increased intracellular IL-8 in monocytes but did not result in detectable cell-surface IL-8.
- IL-1 beta was detected both intracellularly and on the cell surface of LPS-stimulated monocytes.
- ELISA and flow cytometry confirmed quantitative changes in intracellular IL-8 in an LPS-stimulated monocytic cell line.
Conclusions:
- LPS stimulation leads to a time-dependent increase in intracellular IL-8, but not cell-surface IL-8, in monocytes.
- Cell-associated IL-8 is unlikely to have a significant physiological function.
- Flow cytometry is a valuable tool for simultaneously examining cell-surface and intracellular cytokines.
Abstract:
Several cell-associated cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor, exist on the cell surface and are biologically active. Although extracellular IL-8, a potent chemotactic factor for primarily neutrophils, has been studied extensively, cell-associated IL-8 has barely been studied. In this study, we analyzed the intracellular and cell-surface IL-8 in human blood monocytes in vitro by using flow cytometry and predicted the biological activity of the cell-associated IL-8 in vivo. After fixation with paraformaldehyde, mononuclear cells were divided into two subgroups. One subgroup was left untreated to study cell-associated antigens, and the other subgroup was permeabilized with saponin to detect intracellular antigens. In lipopolysaccharide (LPS)-stimulated monocytes, IL-8 was detected solely intracellularly, whereas both the intracellular and cell-surface IL-1 beta was detectable. In a time-course study, the intracellular IL-8 increased in response to LPS stimulation, but the cell-surface IL-8 was undetectable throughout the course. In an LPS-stimulated monocytic cell line, both ELISA and flow cytometry detected the quantitative change of the intracellular IL-8. The dissimilar localization between IL-8 and IL-1 beta within cells was confirmed by the immunohistochemical analysis. In summary, LPS stimulation induced a time-dependent increase in intracellular but not cell-surface IL-8 in monocytes. Thus, it is unlikely that the cell-associated IL-8 is functioning physiologically. The semiquantitative flow cytometric procedure may be useful for simultaneous examination for cell-surface and intracellular cytokines.
